You can read in pET system manual that secondary structure of mRNA transcript can interfere with ATG translation codon or RBS and can lower expression level. I have trouble to express app. 14 kDa bacterial protein, which supposed to be not toxic (al least its homologs from different bacteria are not) – I am using pET28a vector and do not want to add any tag – so I was looking for a reason for not getting any protein (I tried different conditions with temperature or IPTG concentration or cells). Codon usage looks not so bad, but when I did estimation of secondary structure of mRNA using mfold program (found on Prof. Zuker’s web page), I found always some sequences complementary to ATG or RBS. Also with mfold found on another web page I’ve got different results. I’ve already tried to “improve” the secondary structure of mRNA making some silent mutations in regions, which were complementary to ATG or RBS, but it did not improve protein expression. I am still getting nothing. Wha
t do you think about it?
http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0
